A new type of gene-disruption cassette with a rescue gene for Pichia pastoris.

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017.

In vitro selection of a peptide inhibitor of human IL-6 using mRNA display.

Interleukin-6 (IL-6) plays a crucial role in malignant diseases, such as rheumatoid arthritis, Castleman disease, and multiple myeloma, and as such, is an attractive therapeutic target. Here, the authors isolated a novel IL-6 inhibitor peptide by in vitro selection using mRNA display. The authors first used a random-primed human cDNA library to isolate IL-6-binding peptides. After four rounds of selection, a 19-amino acid peptide named CA11 was selected and confirmed to specifically interact with IL-6. The authors then performed an alanine scan analysis of CA11 and determined the amino acid residues necessary to interact with IL-6. Next, the authors constructed a CA11-based partially randomized library and after ten more rounds of selection, isolated several groups of peptides. The most frequently occurring sequence, RA07, bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130.

A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP.

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C.

In vitro selection of scFv and its production: an application of mRNA display and wheat embryo cell-free and E. coli cell production system.

Synthetic DNA libraries encoding human antibody V(L) and V(H) fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the number of molecules used in mRNA display, the size of the library displayed was calculated to be >10(10). To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established.

A completely in vitro system for obtaining scFv using mRNA display, PCR, direct sequencing, and wheat embryo cell-free translation.

Using mRNA display followed by in vitro sequencing and translation, a complete in vitro system for obtaining scFv has been developed. An mRNA display library for synthetic scFv was panned against human TNF receptor (TNFR). The nucleotide portion of the enriched molecules was subjected to limiting dilution, and PCR-amplified. Three of the proteins encoded by the amplified fragments were synthesized in a wheat embryo (WE) cell-free system using a batch method. They were shown to bind TNFR by ELISA. One of their sequences was identified in vitro. The identified clone was further synthesized at approx. 0.5 mg/ml reaction mixture in a WE system with dialysis as a totally soluble protein.

Identification of amino acids essential for antibody binding by mRNA-display using a random peptide library: an anti-human tumor protein p53 antibody as a model.

Using a synthetic DNA library coding for random 10-amino acid peptides (R10aPL), mRNA-display was applied to the isolation of interactive peptides using a monoclonal antibody against human TP53 (hTP53) as a model. Display molecules consisting of peptides and the nucleotide sequences encoding them were synthesized in vitro and subjected to four to five cycles of affinity selection. Thirty-four clones each isolated in the 4th or 5th round were sequenced. A core sequence, (X)-S-D-L-(Z)-K-L essential for binding was found, in which (X) and (Z), though undefined, were mostly F or Y and W, respectively. Although no peptides that fully matched with hTP53 were found in the clones isolated, the core sequence was found in hTP53. A 10-amino acid peptide containing the core sequence was chemically synthesized to verify its binding with SPR. Its Kd value for the antibody was 6 nM. The amino acids in epitopes essential for binding could be identified by mRNA-display with R10aPL.

Isolation of cross-reacting antigen candidates by mRNA-display using a mixed cDNA library.

The identification of cross-reacting antigens is an important process in the characterization of antibodies. We describe here the application of mRNA-display technology with a cDNA library for the isolation of cross-reacting antigens using a monoclonal antibody against human tumor protein p53 (hTP53) as a model. A mixed cDNA library constructed from mRNAs prepared from several human tissues and cell-lines was used for the mRNA-display. After several rounds of panning, five annotated polypeptides, topoisomeraseII-binding protein 1 (TOBP1), RAS protein activator like 2 isoform 1 (RASAL2), endosome-associated FYVE-domain protein (ZFYVE16), and utrophin (UTRN) as well as hTP53, were identified as cross-reactive antigen candidates. All of them had a consensus motif, -S-D-L-( )-K-L-, which was included in the known epitope of the antibody. They were synthesized in vitro, and their binding was compared by conducting a pull-down assay. In cross-activity to the antibody, they ranked as follows: ZYVE16 congruent with hTP53 congruent with TOBP1 > UTRN > RASAL2.